A Systematic Approach to Assess the Activity and Classification of PCSK9 Variants

Background: Gain of function (GOF) mutations of PCSK9 cause autosomal dominant familial hypercholesterolemia as they reduce the abundance of LDL receptor (LDLR) more efficiently than wild-type PCSK9. In contrast, PCSK9 loss of function (LOF) variants are associated with a hypocholesterolemic phenotype. Dozens of PCSK9 variants have been reported, but most remain of unknown significance since their characterization has not been conducted. Objective: Our aim was to make the most comprehensive assessment of PCSK9 variants and to determine the simplest approach for the classification of these variants. Methods: The expression, maturation, secretion, and activity of nine well-established PCSK9 variants were assessed in transiently transfected HEK293 cells by Western blot and flow cytometry. Their extracellular activities were determined in HepG2 cells incubated with the purified recombinant PCSK9 variants. Their binding affinities toward the LDLR were determined by solid-phase immunoassay. Results: LDLR expression increased when cells were transfected with LOF variants and reduced when cells were transfected with GOF variants compared with wild-type PCSK9. Extracellular activities measurements yielded exactly similar results. GOF and LOF variants had increased, respectively reduced, affinities for the LDLR compared with wild-type PCSK9 with the exception of one GOF variant (R218S) that showed complete resistance to inactivation by furin. All variants were expressed at similar levels and underwent normal maturation and secretion patterns except for two LOF and two GOF mutants. Conclusions: We propose that transient transfections of HEK293 cells with a plasmid encoding a PCSK9 variant followed by LDLR expression assessment by flow cytometry is sufficient to reliably determine its GOF or LOF status. More refined experiments should only be used to determine the underlying mechanism(s) at hand.

Role of solid phase immunoassay as a screening test for antinuclear antibody – A comparative analysis on Indian population

Detection of Antinuclear antibody (ANA) is the hallmark of laboratory investigations in Connective Tissue Disorders (CTD). However, various methodologies used in both screening tests and specific antibody detection has led to a loss of consensus and poor reproducibility of results. The objective of this study is to compare Solid Phase Immunoassay (SPI) with Indirect Immunofluorescence (IFA) as a screening test in correlation with the clinical profile as well as subsequent detection of specific antibodies. The study was conducted as a pilot study with a sample size of 60 cases, recruited by Rheumatologists, between April 2019 to July 2019. Each sample was screened by IFA and SPI and tested for specific antibodies by three different specific antibody tests. Although the Sensitivity of SPI (71%) was lower when compared to IFA (79%), the Specificity (78%), Positive Predictive Value (PPV) (74%) and Negative Predictive Value (NPV) (76%) were all comparatively higher. In two clinically proven cases of Sjogren’s syndrome where IFA was negative and SPI was positive, specific antibody tests showed positivity for SSA/Ro. Also it was seen in two clinically confirmed cases of Systemic Lupus Erythematosus IFA was positive and SPI was negative. In this pilot study SPI appeared comparable to IFA as a screening test with better specificity, PPV and NPV. The utility of SPI was especially seen in cases with antibodies against SSA/Ro where IFA may be negative. However, in a few cases of high antibody titer SPI appeared to give a false negative result.

Suppressing Non-Specific Binding of Proteins onto Electrode Surfaces in the Development of Electrochemical Immunosensors

Electrochemical immunosensors, EIs, are systems that combine the analytical power of electrochemical techniques and the high selectivity and specificity of antibodies in a solid phase immunoassay for target analyte. In EIs, the most used transducer platforms are screen printed electrodes, SPEs. Some characteristics of EIs are their low cost, portability for point of care testing (POCT) applications, high specificity and selectivity to the target molecule, low sample and reagent consumption and easy to use. Despite all these attractive features, still exist one to cover and it is the enhancement of the sensitivity of the EIs. In this review, an approach to understand how this can be achieved is presented. First, it is necessary to comprise thoroughly all the complex phenomena that happen simultaneously in the protein-surface interface when adsorption of the protein occurs. Physicochemical properties of the protein and the surface as well as the adsorption phenomena influence the sensitivity of the EIs. From this point, some strategies to suppress non-specific binding, NSB, of proteins onto electrode surfaces in order to improve the sensitivity of EIs are mentioned.

IMMUNOLOGICAL IMBALANCE IN BREAST CANCER AND LUNG CANCER IN POSTMENOPAUSAL WOMEN

Previous studies reported some associations between class A antibodies specific for benzo[a]pyrene (IgA-Bp), estradiol (IgA-Es) and progesterone (IgA-Pg) and breast cancer (BC) in women like as with lung cancer (LC) in men. It was suggested that IgA-Bp and IgA-Es may stimulate tumor initiation and promotion, whereas IgA-Pg may inhibit the in vivo human carcinogenesis.The purpose of this study was to identify the suggested associations of such immunological imbalance with BC and LC in postmenopausal women.The serum A-class antibodies specific to benzo[a]pyrene, estradiol and progesterone (IgA-Bp, IgA-Es, IgA- Pg) were studied in 335 healthy women, 824 breast cancer (BC) patients and 127 cases of lung cancer (LC) by means of non-competitive solid phase immunoassay. The following results were obtained: Increased ratio of IgA-Bp and IgA-Es amounts exceeding the IgA-Pg levels was associated with a higher risk of breast cancer (OR = 2.8 and 2.4 respectively, p < 0.0001), and higher risk of LC (OR = 2.9 and 2.8, respectively, p < 0.0001). Conversely, the OR values decreased to 0.3-0.4 for BC and LC if IgA-Pg levels were higher than IgA-Bp and IgA-Es levels (p < 0.0001). These findings confirm the hypothesis that IgA-Bp and IgA-Es are capable to stimulate, and IgA-Pg, to inhibit the BC and LC occurrence n postmenopausal women. The balance between IgA-Bp and IgA-Es, on the one hand, and IgA-Pg, on the other hand, is much more important than individual contents of these antibodies.In conclusion, the phenomenon of “immunological interference” is revealed, i.e., the mutual enhancement of IgA-Bp and IgA-Es effects, thus, probably, stimulating the initial and subsequent events of carcinogenesis initiation and promotion, with a weak anticancer effect of IgA-Pg, and by weakening the mutual procarcinogenic effects of IgA-Bp and IgA-Es by the marked effect of IgA-Pg.